Structural analysis of a sugar chain of polysaccharide has such a problem that it is difficult to separate and purify a sugar chain. This is caused by the following condition: An amount of a complex sugar chain to be studied is extremely small, and the sugar chain has various types of structures, but sugar itself does not have any marker group.
Conventionally, there has been widely known a method in which a fluorescence labeling group is introduced into a reducing end of a sugar chain of a saccharide so as to confirm analysis or identification of the saccharide with high sensitivity and high accuracy. The inventors of the present invention has developed a pyridylamination method in which a fluorescence labeling group is introduced into the reducing end of the saccharide. As illustrated in FIG. 4, the method is such that 2-aminopyridine serving as the fluorescence labeling group is introduced into the reducing end of the sugar chain so as to obtain the fluorescence-labeled saccharide (pyridylaminated sugar chain).
The analysis of the sugar chain which is carried out by using the pyridylaminated sugar chain has the following advantages.    (1) High sensitivity (detection of 1 fmol amount is possible)    (2) High stability (a pyridylaminated sugar chain prepared 10 years ago has currently no change in fluorescence intensity)    (3) High resolution (a combination with high-performance liquid chromatography (HPLC) allows 1000 sugar chains to be separated)    (4) High versatility (highly compatible with mass spectrometry (MS))    (5) Quantification is possible (only one portion is allowed to enter a single sugar chain)
An example of the conventional pyridylamination method is as follows. First, 20 μl (micro litter: 1 μl corresponds to 10−9 m3) of 2-aminopyridine/acetic acid (552 mg/200 μl) is added to a sample containing a sugar chain, and the resultant is sufficiently stirred, and then is heated at 90° C. for 60 minutes. After the reaction, 70 μl of borane-dimethylamine complex/acetic acid/water (200 mg/80 μl/50 μl) is added thereto, and the resultant is sufficiently stirred, and then is heated at 80° C. for 35 minutes. After the reaction, extraction is carried out by using an organic solvent and foreign matters are removed by using a cation exchange resin, thereby obtaining a pyridylaminated sugar chain.
Further, preparation of a sample containing a sugar chain is carried out by liberating a sugar chain from a glycoprotein or the like. Roughly, there are two methods for liberating a sugar chain. One is a method adopting chemical reaction. The other is a method adopting enzyme reaction.
Herein, hydrazinolysis is described as the method for liberating an N-linked sugar chain from a glycoprotein through chemical reaction. The hydrazinolysis is as follows. As illustrated in FIG. 5 for example, a sample (glycoprotein) is sufficiently dried by lyophilisation or the like and the resultant is mixed with hydrazine anhydride so as to completely dissolve the sample. The resultant is heated at 100° C. for 10 hours. Thereafter, the hydrazine anhydride is distilled under reduced pressure, and a portion having an acetyl group desorped by the hydrazinolysis is acetylated again by using saturated sodium hydrogen carbonate aqueous solution and acetic anhydride, thereby liberating a sugar chain from the glycoprotein.